The Role Of CCL17 In The Cross-presentation Of Long Epitope Peptide-activated CTLs By DCs In Anti-tumor

Objective: This study mainly explores the effect of chemokines CCL17 on the process of long peptides cross-presentation by DC cell.Then we can increase the understanding of the molecular mechanism of cross presentation,and provide new ideas and methods for tumor immunotherapy.Method:In vitro,dendritic cells derived from human peripheral blood mononuclear cells were induced to observe the morphological characteristics of DC cells.The expression of surface markers on DC cells was analyzed by flow cytometry.Immune staining and flow cytometry were used to detect the phagocytic ability of imDC cells to long peptide antigen.ImDC internalized long peptide(Survivin79L)were co-cultured with T lymphocytes.The IFN-?secretion of cytotoxic T lymphocytes induced by peptides in vitro were detected by ELISA.The IFN-?secretion of CTL cells stimulated in vitro were detected when the proteasome inhibitor LLnL added to the cross presentation of DC cells.Calcein-AM were used to test the cytotoxicity of CTL cells induced in vitro against peptide-loaded T2 cells and tumor cells.The expression of chemokine CCL17 in DC cells were detected by RT-PCR and ELISA,respectively.Then ELISA was used to detect the content of chemokine CCL17 in the cross presentation of DC cells.For the further exploration of CCL17 in cross presentation,the phagocytosis ability of imDC cells to long peptide antigen was detected by adding chemokines CCL17 neutralizing antibody or using CRISPR technology,and then the activation ability of imDC cells to CTL cells was detected by ELISA.Calcein-AM were used to test the cytotoxicity of CTL cells induced by different groups against peptide-loaded T2 cells.Then recombinant CCL17 protein was added in cross-presentation of long peptides by imDC cell or in presentation of short peptides by mDC cell.Lastly,the expression of CCL17 receptor CCR4 on CTLs induced by neutralizing antibody in vitro was detected by flow cytometry.Chemotaxis assay was used to detect the chemotaxis ability of CCL17 to two groups of CTLs.Result:ImDC were obtained with larger size and less burr on the surface of the cells on seventh days of culture.The cell surface burr increased and become mature when added TNF-?.And the surface marker molecules CD1 a and CD83 of DC cells were significantly higher than those of imDC cells.ImDC cells have strong phagocytosis to long peptide Survivin79 L.CTLs induced by imDC cells internalized long peptide in vitro and the control group CTLs induced by mDC cells can secrete a mount of IFN-?.The secretion of IFN-? was significantly induced when Survivin79 L long peptide cross-presented by imDCs to CTLs with LLnL.And CTLs in all two groups can lysis peptide-loaded T2 cells and HepG2 cell.The RT-PCR showed that chemokine CCL17 was expressed in DC cells,and the expression of chemokine CCL17 was significantly reduced after the maturation of DCs.Moreover the content of CCL17 in the c?lture supernatant of each group was significantly different.The phagocytic ability of long peptide in imDC cells did not change after adding neutralizing antibody.However,the ability of imDC cells to activate CTL cells decreased after adding neutralizing antibody.Compared with the control group CTLs,the cytotoxicity of CTLs induced by imDC cells added neutralizing antibody in vitro significantly decreased against peptides-loaded T2 cells.Similar res?lts were found in the CCL17-knockout imDC.But the killing ability increased after adding exogenous recombinant CCL17 protein.Moreover,the killing ability also increased after adding the recombinant CCL17 protein to the short peptides-presentation by mDC cells.Compared with the CTL cells without the neutralizing antibody,the expression of CCR4 on the surface of CTLs decreased significantly,and the chemotactic ability of CCL17 also decreased significantly.Conclusion: ImDC cells induced from human peripheral blood mononuclear cells can effectively cross-present long peptide antigen to stimulate CTLs.Chemokine CCL17 can promote the tumor killing capacity of CTLs and the expression of CCR4 molec?les on CTLs.Then chemokine CCL17 can increase the invasion of the CTLs in tumor microenvironment.